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First published online 29 June 2005
doi: 10.1242/dev.01913
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1 Cardiovascular Research Institute, University of California, San Francisco, CA
94143-0130, USA
2 Department of Pediatrics, University of California, San Francisco, CA
94143-0130, USA
3 Genome Sciences Department, Lawrence Berkeley National Laboratory, Berkeley,
CA 94720, USA
4 Department of Biochemistry and Biophysics, University of California, San
Francisco, CA 94143, USA
* Author for correspondence (e-mail: brian.black{at}ucsf.edu)
Accepted 20 May 2005
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: GATA4, GATA, BMP4, BMP, FOXF1, Forkhead, Transcription, Enhancer, Transgenic, Mouse, Liver, Mesenchyme, Lateral mesoderm, Septum transversum
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Tam et al., 2003). Members of
the bone morphogenetic protein (BMP) family, including BMP2, BMP4, BMP5 and
BMP7, are highly expressed in the lateral mesoderm and septum transversum
(Hogan, 1996;
Zhao, 2003), and previous
studies have shown that BMP is necessary to direct the specification of
cardiomyocytes, hepatocytes and gut mesenchyme
(Rossi et al., 2001;
Schultheiss et al., 1997;
Sukegawa et al., 2000;
Zhang et al., 2004). In
response to BMP induction, numerous genes are activated and repressed as cell
fates become increasingly restricted during embryonic development
(Hogan, 1996;
Zhao, 2003).
Members of the Forkhead and GATA families are among the earliest
transcription factors that have been implicated downstream of BMP signaling in
vertebrates and invertebrates (Klinedinst
and Bodmer, 2003; Rossi et
al., 2001; Schultheiss et al.,
1997; Tseng et al.,
2004; Zaffran et al.,
2001). Forkhead domain proteins comprise a large family of
transcription factors that are defined by the presence of a winged-helix
DNA-binding domain, and members of this large superfamily are expressed in
virtually all tissues derived from all three germ layers
(Carlsson and Mahlapuu, 2002).
Among the Forkhead proteins that are restricted to the mesoderm, FOXF1 (FOXF1A
– Mouse Genome Informatics) is a key regulator of embryonic and
extraembryonic mesoderm development, and FOXF1 null embryos die by 9.5 days
post-coitum (dpc) because of defects in the extraembryonic mesoderm
(Kalinichenko et al., 2004;
Kalinichenko et al., 2002;
Mahlapuu et al., 2001a;
Mahlapuu et al., 2001b). Thus,
it is clear that Forkhead proteins, including FOXF1, play crucial roles in the
mesoderm during development. However, the transcriptional pathways downstream
of Forkhead factors in the developing mesoderm remain to be determined.
GATA transcription factors belong to an evolutionarily conserved family of
zinc finger-containing proteins that recognize the consensus DNA sequence
WGATAR (Molkentin, 2000;
Patient and McGhee, 2002).
There are six mammalian GATA factors, which play key roles in gene activation
in multiple lineages including hematopoietic tissues, heart and liver
(Burch, 2005;
Molkentin, 2000;
Shivdasani, 2002;
Weiss and Orkin, 1995;
Zaret, 1999). Among GATA
family members, the Gata4 gene is expressed early in the
post-gastrula embryo and is a key regulator of mesodermal and endodermal
development (Arceci et al.,
1993; Grepin et al.,
1997; Heikinheimo et al.,
1994; Rossi et al.,
2001). Gata4-knockout mice die around 9.5 dpc, and
display defects in heart and foregut morphogenesis that result from severe
defects in the ventral foregut endoderm
(Kuo et al., 1997;
Molkentin et al., 1997;
Narita et al., 1997). GATA4
functions in a variety of transcriptional complexes with multiple other
factors, including NK class homeodomain factors and Forkhead transcription
factors such as FOXA2, to activate downstream genes associated with
specification and differentiation in cardiac mesoderm, prehepatic endoderm and
gut endoderm (Bossard and Zaret,
1998; Denson et al.,
2000; Divine et al.,
2004; Durocher et al.,
1997; Lee et al.,
1998; Sepulveda et al.,
1998; Sepulveda et al.,
2002). However, despite the importance of GATA4 in mouse embryonic
development, the transcription factors upstream of Gata4 have not
been defined.
In this study, we identify for the first time a transcriptional enhancer from the mouse Gata4 gene. This evolutionarily conserved distal enhancer is sufficient to direct expression to the lateral mesoderm in transgenic mouse embryos, beginning at 7.5 dpc. As development proceeds, this novel enhancer element directs robust expression throughout the visceral mesoderm and septum transversum mesenchyme, and, by 11.5 dpc, enhancer activity becomes restricted to the mesenchyme surrounding the liver. We show that the activity of the enhancer is inhibited by the BMP antagonist Noggin in embryo culture explants, and that the enhancer is not active in Bmp4 null embryos, demonstrating that this Gata4 lateral mesoderm enhancer is a downstream target of BMP4. This evolutionarily conserved Gata4 enhancer element contains an essential FOX-binding site that is efficiently bound by the Forkhead transcription factor FOXF1, and this element is required for enhancer activity throughout development. The enhancer also appears to be a target for autoregulation via two perfect consensus GATA sites that are robustly bound by GATA4 and are also required for enhancer function in vivo. Thus, these studies identify Gata4 as a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm. Furthermore, these studies identify Gata4 as a downstream target of BMP4 in the lateral mesoderm and septum transversum, and support a role for GATA4 as a transcriptional mediator of BMP signaling in those lineages.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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).
Deletion fragments of the G2 Gata4 lateral mesodermal enhancer were
generated by digestion using the following restriction sites, followed by
subsequent subcloning back into HSP68-lacZ: G2
1,
SalI-KpnI; G22, PstI; G23,
StuI-XmnI; G24, PstI-StuI.
G25 was generated by PCR using the primers
5'-cactgaggaagcttgtacctgg-3' and
5'-atggatgcctgcagattggtg-3', and subsequent cloning into
HSP68-lacZ as a HindIII-PstI fragment; G26
contains an internal deletion within G2 generated by cleavage with
SphI and religation. Mutations were introduced into the G22
enhancer fragment in HSP68-lacZ for analysis in transgenic mice. The
following mutant sequences were created in the context of fragment G22:
mFox I, 5'-tggggtagtctcgagaagcatcttcagaaaag-3'; m-gata I,
5'-caagacagtagagctcagcagggctc-3'; m-gata II,
5'-ttacaagctccatggaaggcccttgtctttag-3'. To create the double gata
I/gata II mutant, referred to as mGATA, the m-gata II sequence was introduced
into a form of fragment G22 that already contained the m-gata I
sequence. The sequence of each mutant fragment was confirmed by sequencing on
both strands. The SMaa-lacZ reporter construct contains the mouse
smooth muscle 
-actin promoter cloned into plasmid AUG-ß-gal and
has been described previously (Anderson et
al., 2004). The GenBank Accession number for the sequence of the
Gata4 G2 lateral mesoderm enhancer is AY763588.
Generation of transgenic mice and mouse embryo culture
Transgenic reporter fragments were digested from the plasmid backbone with
SalI (fragments G2, G21, G23, G24, G25
and G26), SalI-PstI (G22 and mutants in that
context), or XhoI-SacII (SMaa-lacZ), gel purified,
and suspended in 5 mM Tris-HCl, 0.2 mM EDTA (pH 7.4) at a concentration of 2
µg/ml for pronuclear injection, as described previously
(Hogan et al., 1994). Injected
embryos were implanted into pseudopregnant CD-1 females, and embryos were
collected at indicated times for F0 analysis or were allowed to develop to
adulthood for the establishment of transgenic lines. DNA was extracted from
the yolk sac of embryos, or from tail biopsies from mice by digestion in tail
lysis buffer (100 mM NaCl, 25 mM EDTA, 1% sodium dodecyl sulfate, 10 mM
Tris-Cl, 200 µg/ml of proteinase K, pH 8.0) at 56°C overnight. Digested
samples were extracted once with phenol-chloroform and ethanol precipitated.
The presence of the lacZ transgene was detected either by PCR or
Southern blot. For PCR genotyping, the lacZ primers LACZ5
(5'-cggtgaatggtgctgcgttgga-3') and LACZ3
(5'-accaccgcacgatagagattc-3') were used. For determination of
genotype by Southern blot, DNA samples were digested with SacI,
followed by blotting and hybridization using a radiolabeled lacZ
probe. The Bmp4-knockout mice have been described previously
(Liu et al., 2004).
For embryo explant culture experiments, embryos from a single stable
transgenic line of G2-lacZ or SMaa-lacZ were collected at
9.5 dpc, and the heart, septum transversum and adjacent regions were dissected
and cultured in Dulbecco's modified Eagle's medium supplemented with 1% fetal
bovine serum, penicillin (100 U/ml), streptomycin (100 U/ml) and 2 mM
L-glutamine at 37°C in 5% CO2 in 24-well tissue-culture plates.
Recombinant mouse Noggin (R&D Systems) was prepared in PBS+0.1% BSA and
added to the cultured embryonic tissues at a final concentration of 10 nM for
48 hours. Control tissues were treated with PBS+0.1% BSA without recombinant
Noggin. The hearts continued beating throughout the 48-hour duration of the
experiment. Following treatment, tissues were fixed and X-gal stained for
ß-galactosidase activity, as described previously
(Dodou et al., 2003). All
experiments using animals complied with federal and institutional guidelines,
and were reviewed and approved by the UCSF Institutional Animal Care and Use
Committee.
X-gal staining, immunohistochemistry and in situ hybridization
ß-Galactosidase expression in lacZ transgenic embryos or
tissues was detected by X-gal staining, which was performed as described
previously (Dodou et al.,
2003). Transverse and sagittal sections from X-gal-stained embryos
and tissues were prepared and counterstained with Neutral Fast Red, as
described previously (Anderson et al.,
2004). Whole-mount in situ hybridization was performed as
described previously (Wilkinson and Nieto,
1993). Briefly, embryos were fixed overnight in 4%
paraformaldehyde, then washed twice with phosphate-buffered saline (PBS) and
dehydrated through a series of PBT (1 xPBS+0.1% Tween20)-methanol
washes. Embryos were then rehydrated through a reciprocal series of
PBT-methanol washes and were treated at room temperature with 10 µg/ml
proteinase K for varied times depending on age: 8.25 dpc embryos were
incubated for 1 minute and 9.5 dpc embryos were incubated for 7 minutes. After
proteinase K treatment, embryos were rinsed with 2 mg/ml glycine in PBT
followed by two successive washes in PBT at room temperature. Embryos were
fixed in 4% paraformaldehyde and 0.2% glutaraldehyde for 20 minutes at room
temperature, rinsed three times in PBT, and incubated in hybridization
solution (50% formamide, 1% SDS, 5 xSSC, 50 µg/ml yeast tRNA, 50
µg/ml heparin) for 16 hours at 70°C. Whole-mount in situ hybridization
was carried out with digoxigenin-labeled antisense or sense RNA probes at 100
ng/ml in hybridization buffer.
For in situ hybridization on sections, embryos were fixed, embedded in
paraffin wax, sectioned at a thickness of 7 µm, and dewaxed as described
previously (De Val et al.,
2004). Sections were then digested for 8 minutes in 40 µg/ml
proteinase K, fixed in 4% paraformaldehyde for 20 minutes, dehydrated through
a series of ethanol washes and allowed to dry. In situ hybridization was
performed with digoxigenin-labeled antisense or sense RNA probes at a
concentration of 1 µg/ml in 50 µl of hybridization buffer. Following
hybridization, embryos or sections were washed and treated with RNaseA using
previously described standard methods
(Wilkinson and Nieto, 1993).
Signal was detected using an alkaline phosphatase-conjugated anti-digoxigenin
antibody and BM Purple alkaline phosphatase substrate (Roche Pharmaceuticals).
Following staining, sections were counterstained with Neutral Fast Red. The
Bmp4 in situ probe has been described
(Jones et al., 1991).
Foxf1 antisense probe was generated from pGEM-FOXF1 (HFH-8), which
was kindly provided by R. Costa and has been described
(Peterson et al., 1997).
Gata4 antisense probe was generated from a pBluescript plasmid
containing the mouse Gata4 cDNA sequences from –100 to +350
(relative to the translational start site), linearized with EcoRI and
transcribed with T3 polymerase.
Electrophoretic mobility shift assay (EMSA)
DNA-binding reactions were performed as described previously
(Dodou et al., 2003). Briefly,
double-stranded oligonucleotides were labeled with 32P-dCTP, using
Klenow to fill in the overhanging 5' ends, and purified on a
nondenaturing polyacrylamide-TBE gel. Binding reactions were pre-incubated at
room temperature in 1 xbinding buffer [40 mM KCl, 15 mM HEPES (pH 7.9),
1 mM EDTA, 0.5 mM DTT, 5% glycerol] containing recombinant protein, 1 µg of
poly dI-dC, and competitor DNA for 10 minutes prior to probe addition.
Reactions were incubated for an additional 20 minutes at room temperature
after probe addition and electrophoresed on a 6% nondenaturing polyacrylamide
gel. Foxf1, Foxa2, Gata4, Gata5 and Gata6 cDNAs were
transcribed and translated using the TNT Quick Coupled
Transcription/Translation Systems, as described in the manufacturer's
directions (Promega).
FOXF1 protein was generated from plasmid pCITE-FOXF1. pCITE-FOXF1 was made
by cloning the Foxf1 cDNA from pGEM-FOXF1, which was kindly provided
by R. Costa and has been described
(Peterson et al., 1997), as a
PspOMI insert into the NotI site in pCITE-2A (Novagen). FOXA2 protein
was generated from plasmid pCDNA1-FOXA2, which was made by cloning the mouse
Foxa2 cDNA as an EcoRI-XbaI fragment into the
EcoRI and XbaI sites of pCDNA1/amp (Invitrogen). GATA4
protein was generated from plasmid pCITE-GATA4, which has been described
previously (Dodou et al.,
2004). GATA5 protein was generated from pRK5-GATA5, which was made
by cloning the mouse Gata5 cDNA from pCDNA1-GATA5 as a
EcoRI-XhoI fragment into the EcoRI and
SalI sites in pRK5. GATA6 protein was generated from pCITE-GATA6,
which was made by cloning the mouse Gata6 cDNA from pCDNA1-GATA6 as a
NotI-XbaI fragment into the NotI and XbaI
sites in pCITE-2B (Novagen). The GATA pCDNA1 plasmids were kindly provided by
Jeff Molkentin and have been described previously
(Liang et al., 2001). The
Nkx2.5 gs1 control GATA4-binding site oligonucleotides have been
described (Lien et al., 1999).
The sequences of the control FOXF1- and FOXA2-binding sites have also been
described previously (Overdier et al.,
1994; Peterson et al.,
1997). The sense-strand sequences of the Gata4 G2
oligonucleotides used for EMSA were:
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Because
GATA4 is among the earliest transcription factors expressed in these lineages,
we sought to identify the upstream transcriptional regulation of the
Gata4 gene to define early pathways governing mesoderm and endoderm
development. The expression of Gata4 in multiple germ layers
suggested that it might be regulated by multiple transcriptional enhancers
that each control expression in a single lineage. Therefore, to identify
Gata4 transcriptional enhancers, we compared the sequences of the
mouse and human Gata4 loci for regions of conservation using BLAST
and VISTA analyses (Altschul et al.,
1990; Mayor et al.,
2000). These comparisons identified five regions of strong
conservation in noncoding sequences, referred to as G1-G5, within the
Gata4 locus (Fig. 1).
Based on the notion that conservation occurs preferentially in functionally
important sequences, we tested each of these conserved noncoding sequences by
cloning each into the transgenic reporter plasmid HSP68-lacZ
(Kothary et al., 1989), as
depicted in Fig. 1, and testing
each for activity in transgenic embryos.
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Expression directed by the Gata4 lateral mesoderm enhancer overlaps Foxf1 and Bmp4
To define the Gata4 lateral mesoderm enhancer in more detail, we
compared the expression pattern directed by the enhancer with the expression
pattern of the early mesodermal marker Foxf1
(Peterson et al., 1997). The
expression patterns of the Gata4 transgene and Foxf1
appeared to be nearly identical at 8.25 dpc
(Fig. 2, compare B and D) and
at 9.5 dpc (Fig. 2, compare F and H, and J
and L). Expression directed by the Gata4 G2 mesodermal
enhancer was nearly identical to the expression of Foxf1 in the
septum transversum (Fig. 2, compare J and
L), and was largely, but not completely, overlapping in the gut
mesenchyme (Fig. 2, compare O and
P). By contrast, the gut expression directed by the Gata4
G2 enhancer did not overlap with the gut expression of Foxa2, which
is restricted to the endodermal compartment within the developing gut (not
shown). ß-Galactosidase expression directed by the Gata4
G2-lacZ transgene also overlapped the expression of Bmp4
(Fig. 2, compare F and G, and J and
K), but did not overlap the expression of the hepatic endodermal
marker Hex (not shown).
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A small, evolutionarily conserved module is necessary and sufficient for Gata4 lateral mesoderm enhancer function in vivo
We next wanted to define the minimal region of the Gata4 G2
mesodermal enhancer that was required for expression in vivo. Comparison of
the mouse Gata4 gene sequence with the Gata4 sequence from
the opossum Didelphus virginiana identified three smaller regions of
very high conservation within the G2 sequence, denoted CR1, CR2 and CR3
(Fig. 4A). We reasoned that one
or more of the smaller regions of conservation was likely to be crucial for
enhancer function in vivo because these sequences have been conserved for the
approximately 150 million years since placental and marsupial mammals diverged
(Graves, 1996). Accordingly,
we designed a series of deletion constructs of the G2 region from the mouse
Gata4 gene to define which of these three deeply conserved regions
were important for mesodermal expression at 9.5 dpc
(Fig. 4). The 4368 bp G2
fragment directed robust expression in the septum transversum and visceral
mesoderm at 9.5 dpc (Fig. 4B).
Deletion of the CR2 and CR3 regions from the 3' end of G2 to create
construct G21 (nucleotides 1-3186) completely abolished transgene
activity in vivo (Fig. 4C). By
contrast, a 1358 bp fragment, designated G22 (nucleotides 3011-4368),
that included CR2 and CR3 was sufficient to direct robust transgene expression
in a temporal and spatial pattern that was identical to that of the larger G2
construct (Fig. 4D).
We next created two deletion constructs designed to separate CR2 from CR3,
to determine whether either or both of these regions were sufficient to direct
mesodermal expression in vivo (Fig.
4A). G23 contained only the CR3 region of opossum homology
and was unable to direct any detectable expression in transgenic embryos at
9.5 dpc (Fig. 4E). By contrast,
the G24 construct, which contained the CR2 region but not the other
deeply conserved regions within the G2 region was sufficient to direct the
mesodermal expression pattern observed with the full-length construct
(Fig. 4F). Further dissection
of the CR2 region to create construct G25, a smaller 308 bp region that
contained only the most highly conserved region of sequence within CR2, had a
dramatic and deleterious impact on transgene expression in vivo, although the
pattern of expression appeared similar
(Fig. 4G). Construct
G26, which contained an internal deletion from construct G2 that
removed the CR2 region, was inactive in transgenic embryos
(Fig. 4H). Taken together, the
results of these deletional analyses demonstrate that the CR2 region of
ancient conservation within the Gata4 G2 mesodermal enhancer is
necessary and sufficient for enhancer function in vivo.
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)
comparing the conserved region of the enhancer from mouse, human and opossum
identified perfect conservation in three candidate Forkhead transcription
factor (FOX)-binding sites and three candidate GATA transcription factor
binding sites (Fig. 5). These
analyses also identified two conserved candidate SMAD-binding sites
(Fig. 5). SMAD proteins are
downstream transcriptional effectors of BMP signaling, and, as shown in
Fig. 2, the expression of
Bmp4 largely overlaps with the expression of Gata4
G2-lacZ. Similarly, the expression of the Forkhead transcription
factor gene Foxf1 almost completely overlaps the lacZ
expression directed by the Gata4 G2 enhancer
(Fig. 2). Thus, the putative
SMAD- and FOX-binding sites in the conserved region of the enhancer
represented excellent candidate sites for a potential role in the regulation
of the Gata4 gene. Likewise, the candidate GATA-binding sites in the
enhancer represented excellent potential sites for auto- and cross-regulation
of the enhancer by GATA4 and other GATA family members.
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). FOXF1
bound efficiently to the control FOXF1 site
(Fig. 6B, lane 2) and this
binding was specific, as it was efficiently competed by an excess of unlabeled
control FOXF1 site probe (Fig.
6B, lane 3). Likewise, the binding of FOXF1 to the control FOXF1
site was abolished by the addition of unlabeled Gata4 Fox I site
probe at a 10-fold excess (Fig.
6B, lane 6) but not by a mutant version of the Gata4 Fox
I site, not even when added at a 100-fold excess
(Fig. 6B, lane 7).
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). This binding was specifically abolished by the
addition of excess unlabeled control site
(Fig. 6C, lane 3) and by the
addition of unlabeled G2 Fox I sites (Fig.
6C, lane 4), but not by the addition of unlabeled mutant Fox I
site at a 100-fold excess (Fig.
6C, lane 5). FOXA2 also bound efficiently to the Gata4
Fox I site itself (Fig. 6C,
lane 7), and this binding was efficiently competed away by the addition of
unlabeled FOXA2 control or G2 Fox I site oligonucleotides
(Fig. 6C, lanes 8,9), but not
by a 100-fold excess of the mutant Fox I site
(Fig. 6C, lane 10). FOXA2 is
expressed within the endoderm in the gut, but a recent paper reported that
FOXA2 is also expressed in the mesoderm
(Hu et al., 2004), suggesting
that it or another related Forkhead factor could regulate Gata4
expression in lateral mesoderm. Overall, the results presented in
Fig. 6 demonstrate that the
conserved Fox I site in the Gata4 lateral mesoderm enhancer is a bona
fide binding site for Forkhead transcription factors, including FOXF1.
We also tested the three conserved, putative GATA sites contained within
the CR2 region of the enhancer to determine if they were bound in EMSA by
GATA4, GATA5 or GATA6. The gata I and gata II sites were each efficiently
bound by GATA factors (Fig. 7),
while the gata III site exhibited only very weak binding by GATA factors in
vitro (data not shown). GATA4 bound very robustly to the gata I site in the
Gata4 lateral mesoderm enhancer
(Fig. 7A, lane 2), whereas
GATA5 and GATA6 bound more weakly to that site
(Fig. 7A, lanes 4 and 6,
respectively). Binding to the gata I site by each of the three GATA factors
was specific because the binding of each was efficiently competed by the
addition of excess unlabeled control gata I site
(Fig. 7A, lanes 3,5,7).
Similarly, GATA4 also bound very robustly to the Gata4 G2 enhancer
gata II site (Fig. 7B, lane 2),
whereas the binding of GATA5 and GATA6 to that site was considerably weaker
(Fig. 7B, lanes 4 and 6,
respectively). As with the gata I site, binding of all three factors to the
gata II site was specifically inhibited by the addition of excess unlabeled
gata II probe (Fig. 7B, lanes
3, 5 and 7). Because the binding of GATA4 to both the gata I and gata II sites
appeared to be stronger than GATA5 or GATA6, we examined the binding of GATA4
in additional detail by competing with a bona fide control GATA-binding site
and with mutant versions of the Gata4 gata I and gata II sites
(Fig. 7C). Binding of GATA4 to
the gata I and II sites in the Gata4 enhancer was efficiently
competed in both cases by a 100-fold excess of the Nkx2.5 gs1 control
site (Fig. 7C, lanes 3,8),
which is a bona fide GATA4-binding site and has been described previously
(Lien et al., 1999). The gata
I site was also efficiently competed by excess self probe
(Fig. 7C, lane 4) but not by
mutant version of itself (Fig.
7C, lane 5). Similarly, the gata II site was competed by excess
unlabeled self probe (Fig. 7C,
lane 9) but not by a mutant form of itself
(Fig. 7C, lane 10). These
results demonstrate that the gata I and gata II sites in the Gata4
lateral mesoderm enhancer both represent extremely robust and specific
GATA-binding sites.
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;
von Bubnoff and Cho, 2001), we
tested the ability of SMAD1/SMAD4, SMAD4/SMAD5, SMAD4/SMAD8, and SMAD4 alone
to bind to either of the two candidate SMAD-binding sites in the enhancer. In
no case were we able to detect any binding by SMAD factors to either of the
sites in the enhancer under conditions in which a control SMAD site was bound
by SMAD4 oligomers in EMSA (data not shown). Furthermore, we introduced
mutations predicted to disrupt SMAD protein binding into the two candidate
SMAD elements in the G2 lateral mesoderm enhancer and tested the effect of
those mutations on enhancer function in transgenic embryos. Simultaneous
mutation of both putative SMAD sites in the enhancer had no effect on
transgene expression at any time point examined, including early time points
at 7.75 dpc and 8.5 dpc (data not shown). Taken together, these data suggest
that the two putative elements in the G2 enhancer are not bona fide
SMAD-binding sites and that the Gata4 G2 lateral mesoderm enhancer is
not a direct target of BMP signaling.
Gata4 lateral mesoderm enhancer activity is dependent on Forkhead- and GATA-binding sites
To test the function of the Gata4 FOX and GATA sites in vivo, we
introduced mutations into the Fox I site and both of the functional GATA sites
(gata I and gata II), in the context of the G22-lacZ transgene
(Fig. 4), and determined the
effect of those mutations on enhancer function in transgenic embryos collected
at 7.75 dpc, 9.5 dpc and 11.5 dpc (Fig.
8). The introduced mutations were identical to those used in the
EMSA analyses, which were shown in Figs
6 and
7 to completely ablate FOXF1
and GATA4 binding, respectively. The wild-type G22-lacZ
transgene directed robust expression throughout the lateral mesoderm and
allantois at 7.75 dpc (Fig.
8A). The wild-type construct continued to direct expression
broadly in the visceral mesoderm and septum transversum mesoderm at 9.5 dpc
(Fig. 8D) and directed
expression restricted to mesenchyme surrounding the liver at 11.5 dpc
(Fig. 8G). Mutation of the Fox
I site in the enhancer resulted in a complete disruption of enhancer activity
in 15 out of 15 F0 transgenic embryos at all time points
(Fig. 8B,E,H), indicating a key
role for Forkhead factors in the activation of the enhancer. Likewise, double
mutation of the two bona fide GATA sites in the enhancer completely ablated
enhancer activity in 14 out of 14 independent F0 transgenic embryos at all
time points (Fig. 8C,F,I).
These results demonstrate that Gata4 expression in the lateral
mesoderm is subject to auto- and/or cross-regulation by GATA4 and other GATA
factors.
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).
Furthermore, the broad expression of the G2-lacZ transgene in the
mesoderm and in the allantois, combined with the largely overlapping
expression of Bmp4 in those lineages or in nearby lineages where
signaling could occur from one region to another, prompted us to test whether
BMP4 might be an upstream regulator of Gata4 enhancer activity
(Fig. 9). To test this
hypothesis, we crossed mice harboring a stably integrated G2-lacZ
transgene with Bmp4+/– mice to generate mice that
were G2-lacZ Tg/0; Bmp4+/– and crossed
these mice to Bmp4+/– mice to generate embryos that
were positive for the Gata4 enhancer transgene and null for
Bmp4. Approximately half of the Bmp4-null embryos died prior
to gastrulation because of the role of BMP4 in gastrulation
(Winnier et al., 1995).
However, about half of the Bmp4 null embryos survived gastrulation
and were clearly alive at 7.5 dpc, as has been previously reported
(Winnier et al., 1995). At
this stage, the activity of the Gata4 enhancer was easily detectable
in heterozygous Bmp4 embryos, with strong expression of lacZ
evident in the lateral mesoderm and allantois
(Fig. 9A). By contrast, the
activity of the Gata4 enhancer was barely detectable in the lateral
mesoderm of Bmp4 null embryos
(Fig. 9B), indicating that the
Gata4 lateral mesoderm enhancer requires BMP4 for activity in vivo.
It was difficult to determine the requirement of BMP4 for activation of
Gata4 G2-lacZ in the allantois, as that structure was
missing or severely reduced in Bmp4 null embryos, as has been
reported previously (Winnier et al.,
1995). However, in Bmp4–/– embryos
where some allantoic tissue was present, we never observed transgene
expression (data not shown), suggesting that BMP4 is required for
Gata4 activation in the allantois as well.
|
).
Therefore, we considered the possibility that the severe reduction in
Gata4 transgene expression in Bmp4 null embryos (shown in
Fig. 9B) might be secondary to
an overall mesodermal defect. Although we considered this possibility unlikely
because Bmp4 mutants that survive gastrulation do have embryonic
mesoderm, we wanted to define further the relationship between BMP signaling
and the Gata4 mesodermal enhancer. As an independent approach to
examine the role of BMP signaling on Gata4 enhancer activation, we
tested whether the activity of the enhancer was sensitive to the BMP inhibitor
Noggin.
Transgenic embryos from a single stable G2-lacZ transgenic line
were explanted at 9.5 dpc and the region of the embryos containing the heart,
septum transversum and portions of the visceral mesoderm were maintained in
culture in the presence of BSA (Fig.
9C) or recombinant Noggin (Fig.
9D). As a control, transgenic embryos from a single stable
transgenic smooth muscle -actin line, SMaa-lacZ, which
expresses ß-galactosidase in the heart and somites at 9.5 dpc, were also
explanted in the presence of BSA (Fig.
9E) or Noggin (Fig.
9F). Embryo explants were maintained in culture for 48 hours and
the hearts continued to beat throughout the time course of the experiment,
indicating the explants were viable for the duration of the experiment. A
total of fifteen G2-lacZ transgenic embryo explants from six
different litters were treated with BSA and fifteen were treated with Noggin,
and we compared X-gal staining in the septum transversum of embryo explants
from the two groups. In each case, Noggin-treated Gata4
G2-lacZ transgenic embryos exhibited a clear attenuation of
ß-galactosidase activity (Fig.
9D) when compared with control-treated embryos
(Fig. 9C). By contrast, no
change in ß-galactosidase activity in SMaa-lacZ explants was
observed in the presence of Noggin (Fig.
9F) when compared with BSA-treated control explants
(Fig. 9E), indicating that
embryo explants were not in general crisis as a result of Noggin treatment.
Thus, these results show that the activity of the Gata4 enhancer is
inhibited by Noggin and further demonstrate that Gata4 is a
downstream target of BMP in the septum transversum and lateral mesoderm. Taken
together, the results presented in Fig.
9 indicate that the Gata4 mesodermal enhancer requires
BMP signaling for activation, and suggest that GATA4 may serve as a downstream
effector of BMP4 in the lateral mesoderm and septum transversum
mesenchyme.
|
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Heikinheimo et al., 1994;
Nemer and Nemer, 2003), and
targeted inactivation of the Gata4 gene in mice results in delayed
ventral morphogenesis and subsequent cardia bifida as a result of severe
endodermal defects (Kuo et al.,
1997; Molkentin et al.,
1997; Narita et al.,
1997). However, despite the importance of Gata4 during
development, it has been unclear how Gata4 expression is activated.
In this manuscript, we identified the first transcriptional enhancer from the
mouse Gata4 gene, and we show that this novel enhancer is active
throughout the lateral mesoderm beginning at 7.5 dpc. As development proceeds,
expression directed by the Gata4 lateral mesoderm enhancer becomes
successively restricted to the visceral mesoderm and to the mesodermal
component of the septum transversum (Fig.
2), and finally becomes restricted to the mesenchyme surrounding
the liver at 11.5 dpc (Fig.
3). The Gata4 lateral mesoderm enhancer requires an evolutionarily conserved Forkhead-binding site for activity in vivo (Fig. 8), and FOXF1 binds to the Forkhead-binding site in the enhancer with high affinity (Fig. 6). Thus, these results demonstrate that Gata4 is a direct transcriptional target of Forkhead factors, and we believe that FOXF1 is a likely candidate based on the overlapping expression pattern of Foxf1 and the G2-lacZ transgene (Fig. 2). The Gata4 lateral mesoderm enhancer also requires two high-affinity GATA-binding sites for function in vivo (Fig. 8), suggesting an essential role for auto- and cross-regulation of Gata4 expression by itself and other GATA factors.
GATA4 as a downstream effector of BMP signaling
BMP proteins are key mediators of tissue specification and of patterning in
multiple developmental lineages. BMPs are known to play essential roles in
bone and cartilage development, neural patterning, heart development,
development of the extraembryonic mesoderm and in signaling from the mesoderm
to the endoderm during endodermal specification
(Hogan, 1996;
Shivdasani, 2002;
Tam et al., 2003;
Zaret, 2001;
Zhao, 2003). As members of the
TGFß superfamily of signaling molecules, BMP signaling modulates gene
expression, at least in part, through the action of SMAD transcription factors
(Derynck and Zhang, 2003;
von Bubnoff and Cho, 2001). It
has been proposed that BMP factors are able to exert such a diverse array of
downstream effects through the interactions of SMAD proteins with other
transcription factors and through the resulting downstream activation of
numerous transcription factor networks
(Derynck and Zhang, 2003). In
this regard, several studies have suggested that GATA transcription factors
are downstream targets of BMP signaling. In response to BMP signaling,
Gata4 expression is upregulated in the precardiac mesoderm in the
chick (Schultheiss et al.,
1997). Similarly, BMP4 signaling from the mesoderm positively
induces the expression of Gata4 in prehepatic endoderm during liver
development, demonstrating activation of Gata4 by BMP4 over a range
from mesoderm to endoderm (Rossi et al.,
2001). However, the pathways through which Gata4 is
activated in response to BMP4 have not been defined in the endoderm or the
mesoderm. Here, we show that the activity of the Gata4 lateral
mesoderm enhancer is attenuated by the BMP antagonist Noggin and is
significantly inhibited in the absence of BMP4
(Fig. 9). Taken together with
our analyses of the SMAD-binding sites in the enhancer, these observations
demonstrate that Gata4 is probably an indirect downstream target of
BMP4 in the lateral mesoderm via the enhancer described here, and suggest that
GATA4 functions as a downstream effector of BMP signaling in the mesoderm.
A reinforcing transcriptional pathway dependent on GATA4
In this study, we demonstrate that the Gata4 lateral mesoderm
enhancer requires an essential Forkhead-binding site that is efficiently bound
by FOXF1 (Figs 6,
8), supporting the possibility
that BMP activation of Gata4 could be mediated by FOXF1 or other
Forkhead proteins. Indeed, a recent study performed in Xenopus
embryos has shown that Foxf1 expression is reduced when BMP4
signaling is reduced, indicating that FOXF1 is also a downstream target of
BMP4 (Tseng et al., 2004).
Interestingly, however, Bmp4 has been shown to be a potential target
of FOXF1 during mouse development, as the level of Bmp4 mRNA was
significantly reduced in the posterior primitive streak, the lateral plate and
the allantois in FOXF1 null embryos
(Mahlapuu et al., 2001b).
Taken together, these results suggest that FOXF1 and BMP4 may function in a
reinforcing regulatory circuit designed to amplify a transcriptional response
downstream of BMP4 signaling, possibly via GATA and other transcription factor
families. GATA transcription factors also appear to activate Bmp4
expression, further supporting the notion of a reinforcing transcriptional
network (Nemer and Nemer,
2003; Peterkin et al.,
2003). The mouse and Xenopus Bmp4 regulatory sequences
contain GATA-binding sites that are functional in studies performed in vitro,
suggesting that Bmp4 expression may be activated or maintained by
GATA factors (Nemer and Nemer,
2003; Peterkin et al.,
2003). A reinforcing transcriptional model is consistent with the
work presented here, which suggests a model for Gata4 activation in
the lateral mesoderm in which BMP4 activates the expression of Gata4
indirectly via FOXF1, and GATA4 functions in a positive-feedback loop to
reinforce its own expression through essential GATA sites in its enhancer
(Fig. 10).
Modular regulation of Gata4 transcription
During embryonic development, Gata4 is expressed in multiple
tissues and exhibits a dynamic and distinct pattern of expression in each
different lineage where it is expressed
(Arceci et al., 1993;
Heikinheimo et al., 1994;
Parmacek and Leiden, 1999).
Notably, the Gata4 enhancer described here directs expression only to
a subset of the endogenous pattern of Gata4 expression, suggesting
that other distinct modular enhancers sufficient to direct expression to other
lineages are also likely to be present within the Gata4 locus. This
type of modular regulation has been observed for other key transcriptional
regulators of tissue specification and differentiation, including
Nkx2.5 (Schwartz and Olson,
1999) and mef2c (De
Val et al., 2004; Dodou et
al., 2004; Dodou et al.,
2003), and modular regulation has been suggested as a mechanism
for regulatory diversity (Firulli and
Olson, 1997). Given the role of GATA4 as an early regulator of
cardiac and endodermal development, it will be important to identify enhancers
sufficient to direct expression to those lineages also. It will be
particularly interesting to determine whether Gata4 is a target of
Forkhead proteins and is subject to auto-regulation in other lineages, as it
is in the lateral mesoderm, or whether regulation in other lineages is via
distinct transcriptional hierarchies.
|
While the importance of GATA4 during embryogenesis is clearly established,
an understanding of its transcriptional regulation has remained elusive. Here,
we show that the expression of Gata4 in the lateral mesoderm and
septum transversum is mediated by an enhancer located 
40 kb upstream of
the start of transcription. Detailed analyses of this enhancer show functional
FOX and GATA sites that are essential for transcriptional activation in vivo.
These data suggest that transcriptional regulation of Gata4 is
modular, and that enhancer elements may be remote from the coding sequences of
the gene. The use of deep evolutionary sequence conservation allowed the
identification of its early mesodermal enhancer and suggests that enhancers
driving cardiac and endodermal expression might be identified through a
similar process. Indeed, we have recently identified a separate enhancer from
the Gata4 gene that is sufficient to direct expression to the
endoderm (A.R. and B.L.B., unpublished). It will be interesting to determine
whether pathways similar to those described here also regulate Gata4
expression in the endoderm and other lineages where it is expressed.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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